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WO2000071162A2 - Nouveaux bioconjugues cyanine et indocyanine pour application medicale - Google Patents

Nouveaux bioconjugues cyanine et indocyanine pour application medicale Download PDF

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WO2000071162A2
WO2000071162A2 PCT/US2000/011060 US0011060W WO0071162A2 WO 2000071162 A2 WO2000071162 A2 WO 2000071162A2 US 0011060 W US0011060 W US 0011060W WO 0071162 A2 WO0071162 A2 WO 0071162A2
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WO2000071162A3 (fr
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Samuel Achilefu
Richard Bradley Dorshow
Joseph Edward Bugaj
Raghavan Rajagopalan
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Mallinckrodt Inc
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Mallinckrodt Inc
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Priority to DE60042522T priority Critical patent/DE60042522D1/de
Priority to DK00926343T priority patent/DK1178830T3/da
Priority to AT00926343T priority patent/ATE435662T1/de
Priority to CA2373475A priority patent/CA2373475C/fr
Priority to JP2000619463A priority patent/JP2003500367A/ja
Priority to AU44888/00A priority patent/AU4488800A/en
Priority to EP00926343A priority patent/EP1178830B1/fr
Publication of WO2000071162A2 publication Critical patent/WO2000071162A2/fr
Publication of WO2000071162A3 publication Critical patent/WO2000071162A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence

Definitions

  • This invention relates generally to novel dye-bioconjugates for use in diagnosis and therapy.
  • this invention relates to novel compositions of cyanine dye bioconjugates of bioactive molecules for site-specific delivery of these agents for optical tomographic, endoscopic, photoacoustic, sonofluorescent, laser assisted guided surgery, and therapeutic purposes. More particularly, this invention relates to a method of preparation and use of cyanine dye bioconjugates for visualization and detection of tumors. This invention is also related to the method of preventing fluorescence quenching by the use of biocompatible organic solvents.
  • the present invention relates particularly to the novel composition
  • novel composition comprising cyanine dye bioconjugates of general formula 1 wherein a and b vary from 0 to 5; W 1 and X 1 may be
  • Formula 1 the same or different and are selected from the group consisting of -CR 10 R n , -O-, -NR 12 , -S-, or -Se;
  • Q 1 is a single bond or is selected from the group consisting of -O-, -S-, -Se-, and -NR 13 ;
  • R 1 , R 10 to R 15 , and R 29 -R 40 may be the same or different and are selected from the group consisting of hydrogen, C,-C 10 alkyl, C,-C 10 aryl, C,-C 10 alkoxyl, C,-C, 0 polyalkoxyalkyl, -CH 2 (CH 2 OCH 2 ) c -CH 2 - OH, C r C 20 polyhydroxyalkyl, C r C 10 polyhydroxyaryl, C,-C 10 aminoalkyl, -(CH 2 ) d -CO 2 H, -(CH 2 ) e - CONH-Bm, -CH 2 -(CH 2 OCH 2 ) r CH 2 -CONH-Bm, -(CH 2 ) g -NHCO-Bm, -CH 2 -
  • NHCO-Bm -(CH ⁇ -OH or -CH 2 -(CH 2 OCH 2 ) zz -CH 2 -OH;
  • Y 1 is selected from the group consisting of -(CH 2 ),-CONH-Bm, -CH 2 -(CH 2 OCH 2 ).-CH 2 -CONH-Bm, -(CH 2 ) k -NHCO-Bm, -CH 2 -(CH 2 OCH 2 ) r CH 2 -NHCO-Bm, -(CH 2 ) m -N(R 14 )-(CH 2 ) n -CONH-Bm, (CH 2 ) aa -N(R 29 )-(CH 2 ) bb -NHCO-Bm, -(CH 2 ) p - N(R I5 )-CH 2 -(CH 2 OCH 2 ) q -CH 2 -CONH-Bm, -(CH 2 ) cc
  • NHCO-Bm -CH 2 -(CH 2 OCH 2 ) complicat-CH 2 -N(R 33 )-CH 2 -(CH 2 OCH 2 ) JJ -CH 2 -CONH-Bm or -CH 2 - (CH 2 OCH 2 ) kk -CH 2 -N(R 34 )-CH 2 -(CH 2 OCH 2 ) 11 -CH 2 -NHCO-Bm; d, e, g, i, k, m, n, p, aa, bb, cc, ff, hh and yy vary from 1 to 10; c, f. h. j, 1, q, dd.
  • Bm is any bioactive peptide, protein, cell, oligosaccharide, glycopeptide, peptidomimetic, drug, drug mimic, hormone, metal chelating agent, radioactive or nonradioactive metal complex, or echogenic agent
  • Z 1 is selected from the group consisting of -(CH 2 ) r -CO 2 H, -(CH 2 ) r -OH, -(CH 2 ) r -NH 2 , -CH 2 - (CH 2 OCH 2 ) s -CH 2 -CO 2 H, -CH 2 -(CH 2 OCH 2 ) s -CH 2 -OH, -CH 2 -(CH 2 OCH 2 ).-CH 2 -NH 2 , -(CH 2 ) t -CONH-
  • R 2 to R 9 may be the same or different and are selected from the group consisting of hydrogen, C,-C ⁇ 0 alkyl, C,-C ]0 aryl, hydroxyl, C,-C ]0 polyhydroxyalkyl, C,-
  • the present invention also relates to the novel composition comprising cyanine dye bioconjugates of general formula 2 wherein a' and b' are defined in the same manner as a and b; W 2 and X 2 are defined in the same manner W 1 and X 1 ; Q 2 is defined in the same manner as Q 1 ; R 16 is defined in the same manner as R 1 ; Y 2 is defined in the same manner as Y 1 ; Z 2 is defined in the same manner as Z 1 ; and R' 7 to R 28 are defined in the same manner as R 2 .
  • This invention is also related to the method of preventing fluorescence quenching. It is known that cyanine dyes generally form aggregates in aqueous media leading to fluorescence quenching. This problem is further accentuated by the conjugation of large hydrophobic dyes to small molecular peptides. We observed that the addition of a biocompatible organic solvent such as 1-50% dimethylsulfoxide (DMSO) restored the fluorescence by preventing aggregation and allowed the visualization of tumors.
  • DMSO dimethylsulfoxide
  • the dye-peptide conjugates are useful for optical tomographic, endoscopic, photoacoustic and sonofluorescent applications for the detection and treatment of tumors and other abnormalities.
  • the dye-peptide conjugates of the invention are useful for localized therapy.
  • the dye peptide conjugates of the invention are useful for the detection of the presence of tumors and other abnormalities by monitoring the blood clearance profile of the conjugates.
  • the dye-peptide conjugates are useful for laser assisted guided surgery for the detection of small micrometastases of, e.g., somatostatin subtype 2 (SST-2) positive, tumors upon laparoscopy.
  • the dye-peptide conjugates of this invention are useful for diagnosis of atherosclerotic plaques and blood clots.
  • Figures 1A-F represent images at 2 minutes and 30 minutes post injection of indocyanine green into rats with various tumors.
  • Figures 1A-B are images of a rat with an induced pancreatic ductal adeno carcinoma tumor (DSL 6A) imaged at 2 minutes ( Figure 1 A) and 30 minutes (Figure IB) post injection.
  • Figures 1C-D are images of a rat with an induced prostatic carcinoma tumor (R3327-H) imaged at 2 minutes ( Figure 1C) and 30 minutes (Figure ID) post injection.
  • F are images of a rat with an induced pancreatic acinar carcinoma (CA20948) expressing the SST-2 receptor imaged at 2 minutes ( Figure IE) and 30 minutes ( Figure IF) post injection.
  • Figures 2A-B show a comparison of the uptake of indocyanine green and Cytate 1 at 45 minutes post injection in rats with the pancreatic acinar carcinoma (CA20948).
  • Figures 3A-B show images of rats with the pancreatic acinar carcinoma (CA20948) 90 minutes ( Figure 3 A) and 19 hours (Figure 3B) post injection of Cytate 1.
  • Figures 4A-B show images comparing selected tissue parts of a rat with the pancreatic acinar carcinoma (CA20948) 22 hours post injection with Cytate 1 ( Figure 4 A) and the same tissue parts imaged in an uninjected rat ( Figure 4B).
  • Figure 5 is an image of individual organs taken from a rat with pancreatic acinar carcinoma
  • Figure 6 is the clearance profile of Cytate 1 from the blood of a normal rat monitored at 830 nm after excitation at 780 nm.
  • Figure 7 is the clearance profile of Cytate 1 from the blood of a CA20948 tumored rat monitored at 830 nm after excitation at 780 nm.
  • Figure 8 is the clearance profile of Cytate 2 from the blood of a normal rat monitored at 830 nm after excitation at 780 nm.
  • Figure 9 is the clearance profile of Cytate 2 from the blood of a CA20948 tumored rat monitored at 830 nm after excitation at 780 nm.
  • Figure 10 is the clearance profile of Cytate 4 from the blood of a normal rat monitored at 830 nm after excitation at 780 nm.
  • novel bioconjugates of the present invention exploit the symmetric nature of the cyanine and indocyanine dye structures by incorporating one to ten receptor targeting groups, in close proximity to each other such that the receptor binding can be greatly enhanced due to a cooperative effect. Accordingly, several cyanine dyes containing one or more targeting domains have been prepared and tested in vivo for biological activity.
  • novel compositions of the present invention comprise dye-bioconjugates of formulas 1 and 2 and offer significant advantages over those currently described in the art. They are applicable to various biomedical applications including, but not limited to, tomographic imaging of organs; monitoring of organ functions; coronary angiography; fluorescence endoscopy; detection, imaging, and therapy of tumors; laser guided surgery, photoacoustic and sonofluorescent methods; and the like. Specific embodiments to accomplish some of the aforementioned biomedical applications are given below.
  • the bioconjugates are prepared by the methods well known in the art and are shown in Schemes (1-3).
  • the bioconjugates according to the present invention have the general formula 1 wherein a and b vary from 0 to 3; Q 1 is a single bond; R 1 to R 9 are hydrogens; W 1 and X 1 are -CR 10 R ⁇ ; Y 1 is -(CH 2 ),-CONH-Bm, -(CH 2 ) k -NHCO-Bm, or -(CH 2 ) m -N(R 14 )-(CH 2 ) n - CONH-Bm; Z 1 is -(CH 2 ) r -OH, -(CH 2 ) r -CO 2 H, -(CH 2 ) r -NH 2 , -(CH 2 ) t -CONH-Dm, -(CH 2 ) v -NHCO- Dm, -(CH 2 ) w -N(R 14 )-(CH 2 ) x -CONH-Dm, -CH 2
  • Bm is a tumor specific biomolecule or drug mimic selected from the group consisting of peptides or oligosaccharides containing 2-50 monomer units and including somatostatin, bombesin, neurotensin, cholecystokinin and vasoactive intestinal polypeptide.
  • the bioconjugates according to the present invention have the general formula 1 wherein a and b are 3; Q 1 is a single bond; R 1 to R 9 are hydrogens; W 1 and X 1 are -C(CH 3 ) 2 ; Y 1 is -(CH 2 ),-CONH-Bm or -CH 2 -(CH 2 OCH 2 ),-CH 2 -CONH-Bm wherein i varies from 1 to 4; and Z 1 is -(CH 2 ) r -CO 2 H, -(CH 2 ) t -CONH-Dm, -CH 2 -(CH 2 OCH 2 ) s -CH 2 -CO 2 H or -CH 2 - (CH 2 OCH 2 ) u -CH 2 -CONH-Dm, wherein r and t vary from 1-4; and Bm is a tumor specific biomolecule selected from the group consisting of Octreotate and its mimics, Octreotide derivatives and
  • the bioconjugates according to the present invention have the general formula 2 wherein a' and b' vary from 0 to 3; Q 2 is a single bond; R' 6 to R 28 are hydrogens; W 2 and X 2 are -CR'°R"; Y 2 is -(CH 2 ),-CONH-Bm, -(CH 2 ) k -NHCO-Bm, or -(CH 2 ) m - N(R 14 )-(CH 2 ) n -CONH-Bm; Z 2 is -(CH 2 ) r -CO 2 H, -(CH 2 ) r -NH 2 , -(CH 2 ) r -OH, -(CH 2 ) t -CONH-Dm, - (CH 2 ) -NHCO-Dm, -(CH 2 ), v -N(R 14 )-(CH 2 ⁇ -CONH-Dm, -CH 2 -(CH
  • Bm is a tumor specific biomolecule or drug mimic selected from the group consisting of peptides and oligosaccharides containing 2-50 monomer units.
  • the bioconjugates according to the present invention have the general formula 2 wherein a and b' are 3; Q 2 is a single bond; R 16 to R 28 are hydrogens; W 2 and X 2 are -C(CH 3 ) 2 ; Y 2 is -(CH 2 ),-CONH-Bm or -CH 2 -(CH 2 OCH 2 ),-CH 2 -CONH-Bm wherein i varies from 1 to 4; and Z 2 is -(CH 2 ) r -CO 2 H, -(CH 2 ) r CONH-Dm, -CH 2 -(CH 2 OCH 2 ) s -CH 2 -CO 2 H or -CH 2 - (CH 2 OCH 2 ) u -CH 2 -CONH-Dm, wherein r and t vary from 1-4; and Bm is a tumor specific biomolecule selected from the group consisting of Octreotate derivatives and their mimics,
  • Octreotide derivatives and their mimics bombesin analogs and their mimics, cholecystokinin analogs and their mimics, and neurotensin analogs and their mimics.
  • a therapeutic procedure comprises attaching a porphyrin to a bioconjugate and using it for photodynamic therapy or shining light of a specific wavelength on the dipeptide conjugate of this invention to achieve a photodynamic therapy effect.
  • compositions of the invention can be formulated into diagnostic compositions for enteral or parenteral administration.
  • These compositions contain an effective amount of the dye along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated.
  • parenteral formulations advantageously contain a sterile aqueous solution or suspension of dye according to this invention.
  • Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration.
  • Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride.
  • Formulations for enteral administration may vary widely, as is well known in the art. In general, such formulations are liquids which include an effective amount of the dye in aqueous solution or suspension. Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like. Compositions for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities.
  • the diagnostic compositions are administered in doses effective to achieve the desired enhancement.
  • doses may vary widely, depending upon the particular dye employed, the organs or tissues which are the subject of the imaging procedure, the imaging equipment being used, and the like.
  • compositions of the invention are used in the conventional manner.
  • the compositions may be administered to a patient, typically a warm-blooded animal, either systemically or locally to the organ or tissue to be imaged, and the patient then subjected to the imaging procedure.
  • a combination of the above represents an important approach to the use of small molecular targeting groups to image tumors by the optical methods.
  • the present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
  • the octapeptide was prepared by an automated fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis using a commercial peptide synthesizer from Applied Biosystems (Model 432 A SYNERGY Peptide Synthesizer).
  • the first peptide cartridge contained Wang resin pre-loaded with Fmoc-Thr on 25 ⁇ mole scale.
  • Subsequent cartridges contained Fmoc-protected amino acids with side chain protecting groups for the following amino acids: Cys(Acm), Thr(t-Bu), Lys(Boc), Trp(Boc) and Tyr(t-Bu).
  • the amino acid cartridges were placed on the peptide synthesizer and the product was synthesized from the C- to the N-terminal position.
  • the coupling reaction was carried out with 75 ⁇ moles of the protected amino acids in the presence of 2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt).
  • HBTU 1,3,3- tetramethyluronium hexafluorophosphate
  • HOBt N-hydroxybenzotriazole
  • the Fmoc protecting group was removed with 20% piperidine in dimethylformamide.
  • the thiol group was cyclized with thallium trifluoroacetate and the product was cleaved from the solid support with a cleavage mixture containing trifluoroacetic acid (85%):water (5%):phenol (5%):thioanisole (5%) for 6 hours.
  • the peptide was precipitated with t-butyl methyl ether and lyophilized with water: acetonitrile (2:3) mixture.
  • the peptide was purified by HPLC and analyzed with LC/MS.
  • the amino acid sequence of Octreotate is: D-Phe-Cys'-Tyr-D-Trp-Lys-Thr- Cys'-Thr (SEQ ID NO:l), wherein Cys' indicates the presence of an intramolecular disulfide bond between two cysteine amino acids.
  • Octreotide was prepared by the same procedure : D-Phe-Cys'-Tyr-D-Trp-Lys-Thr-Cys'-Thr- OH (SEQ ID NO:2), wherein Cys' indicates the presence of an intramolecular disulfide bond between two cysteine amino acids.
  • Bombesin analogs were prepared by the same procedure except that cyclization with thallium trifluoroacetate was not needed. Side-chain deprotection and cleavage from the resin was carried out with 50 ⁇ L each of ethanedithiol, thioanisole and water, and 850 ⁇ L of trifluoroacetic acid .
  • Cholecystokinin octapeptide analogs were prepared as described for Octreotate without the cyclization step. Three analogs were prepared: Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH 2 (SEQ ID NO:5); Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH 2 (SEQ ID NO:6); and D-Asp-Tyr-Nle-Gly-Trp-Nle- Asp-Phe-NH 2 (SEQ ID NO:7) wherein Nle is norleucine. Neurotensin analog was prepared as described for Octreotate without the cyclization step:
  • EXAMPLE 7 Synthesis of Peptide-Dye Conjugates The method described below is for the synthesis of Octreotate conjugates but a similar procedure is used for the synthesis of other peptide-dye conjugates.
  • the Octreotate was prepared as described in Example 6 but the peptide was not cleaved from the solid support and the N-terminal Fmoc group of Phe was retained. The thiol group was cyclized with thallium trifluoroacetate and the Phe was deprotected to liberate the free amine. Bisethylcarboxymethylindocyanine dye (53 mg, 75 ⁇ moles) was added to an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO (375 ⁇ L), and 0.2 M solution of diisopropylethylamine in DMSO (375 ⁇ L).
  • Octreotate-bispentylcarboxymethylindocyanine dye was prepared as described above with some modifications.
  • Bispentylcarboxymethylindocyanine dye (60 mg, 75 ⁇ moles) was added to an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO (400 ⁇ L), and 0.2 M solution of diisopropylethylamine in DMSO (400 ⁇ L).
  • the activation was complete in about 30 minutes and the resin-bound peptide (25 ⁇ moles) was added to the dye.
  • the reaction was carried out at room temperature for 3 hours.
  • the mixture was filtered and the solid residue was washed with DMF, acetonitrile and THF.
  • the peptide was cleaved from the resin and the side chain protecting groups were removed with a mixture of 85% trifluoroacetic acid, 5% water, 5% thioanisole and 5% phenol.
  • the resin was filtered and cold t-butyl methyl ether (MTBE) was used to precipitate the dye-peptide conjugate which was dissolved in acetonitrile:water (2:3) mixture and lyophilized.
  • MTBE cold t-butyl methyl ether
  • the product was purified by HPLC to give Octreotate- 1,1, 2-trimethyl- [lH]-benz[e]indole propanoic acid conjugate (10%), monoOctreotate- bispentylcarboxymethylindocyanine dye (Cytate 3, 60%) and bisOctreotate- bispentylcarboxymethylindocyanine dye (Cytate 4, 30%).
  • the dye-peptide conjugates are sparingly soluble in water and require the addition of solubilizing agents or co-solvents. Addition of 1-20% aqueous ethanol to the conjugates partially quenched the fluorescence intensity in vitro and the fluorescence was completely quenched in vivo (the conjugate was not detected by the CCD camera). Addition of 1-50% of DMSO either re- established or increased the fluorescence intensity of the conjugates in vitro and in vivo. The dye fluorescence remained intense for over one week. The DMSO formulations were well tolerated by experimental animals used for this invention.
  • a non-invasive in vivo fluorescence imaging apparatus was employed to assess the efficacy of contrast agents developed for tumor detection in animal models.
  • a LaserMax Inc. laser diode of nominal wavelength 780 nm and nominal power of 40 mW was used.
  • the detector was a Princeton Instruments model RTE/CCD-1317-K/2 CCD camera with a Rodenstock 10 mm F2 lens (stock #542.032.002.20) attached.
  • An 830 nm interference lens (CVI Laser Corp., part # Fl 0-830-4-2) was mounted in front of the CCD input lens such that only emitted fluorescent light from the contrast agent was imaged.
  • an image of the animal was taken pre-injection of contrast agent. This image was subsequently subtracted (pixel by pixel) from the post injection images.
  • the background subtraction was never done once the animal had been removed from the sample area and returned at a later time for images taken several hours post injection.
  • DSL 6A tumors were induced in male Lewis rats in the left flank area by the introduction of material from a solid (donor) implant and the tumors were palpable in approximately 14 days.
  • the animals were anesthetized with rat cocktail (xylazine; ketamine; acepromazine 1.5: 1.5: 0.5) at 0.8 mL/kg via intramuscular injection.
  • the area of the tumor (left flank) was shaved to expose tumor and surrounding surface area.
  • a 21 gauge butterfly equipped with a stopcock and two syringes containing heparinized saline was placed into the later tail vein of the rat. Patency of the vein was checked prior to administration of the ICG via the butterfly apparatus.
  • Each animal received 500 ⁇ L of a 0.42 mg/niL solution of ICG in water.
  • the images obtained at 2 and 30 minutes post injection are shown in Figures 1A-B.
  • Example 9 The imaging apparatus and the procedure used are described in Example 9.
  • R3327-H tumors were induced in young male Copenhagen rats in the left flank area from a solid implant. These tumors grow very slowly and palpable masses were present 4-5 months post implant.
  • the images obtained at 2 and 30 minutes post injection are shown in Figure 1C-D.
  • EXAMPLE 11 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948 with Indocyanine Green (ICG) The imaging apparatus and the procedure used are described in Example 9.
  • Rat pancreatic acinar carcinoma expressing the SST-2 receptor (CA20948) were induced by solid implant technique in the left flank area and palpable masses were detected 9 days post implant. The images obtained at 2 and 30 minutes post injection are shown in Figure 1E-F.
  • EXAMPLE 12 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) with Cytate 1 The imaging apparatus and the procedure used are described in Example 9 except that each animal received 500 ⁇ L of a 1.0 mg/mL solution of Cytate 1 solution of 25% dimethylsulfoxide in water.
  • Rat pancreatic acinar carcinoma expressing the SST-2 receptor (CA20948) were induced by solid implant technique in the left flank area and palpable masses were detected 24 days post implant. Images were obtained at various times post injection. Uptake into the tumor was seen at 2 minutes but was not maximal until about 5 minutes. Figures 2A-B show a comparison of the uptake of ICG and Cytate 1 at 45 minutes in rats with the CA20948 tumor cell line. By 45 minutes the ICG has mostly cleared ( Figure 2 A) whereas the Cytate 1 is still quite intense ( Figure 2B). This dye fluorescence remained intense in the tumor for several hours post-injection.
  • ICG indocyanine green
  • DSL 6/A pancreatic
  • Dunning R3327-H prostate
  • CA20948 pancreatic
  • the first two tumor lines are not as highly vascularized as CA20948 which is also rich in somatostatin (SST-2) receptors. Consequently, the detection and retention of a dye in this tumor model is an important index of receptor-mediated specificity.
  • Octreotate is known to target somatostatin (SST-2) receptors, hence, we prepared cyano- Octreotates (Cytate 1 and Cytate 2). Cytate 1 was evaluated in the CA20948 Lewis rat model. Using the CCD camera apparatus strong localization of this dye was observed in the tumor at 90 minutes post injection ( Figure 3 A). At 19 hours post injection the animal was again imaged (Figure 3B) and tumor visualization was easily observed showing specificity of this agent for the SST-2 receptors present in this tumor line. As a control, the organs were imaged again (Figure 4A) and the image was compared with that of the same tissues in the uninjected rat ( Figure 4B). Individual organs were removed and imaged.
  • SST-2 somatostatin
  • EXAMPLE 14 Monitoring of the blood clearance profile of peptide-dye conjugates A laser of appropriate wavelength for excitation of the dye chromophore was directed into one end of a fiber optic bundle and the other end was positioned a few millimeters from the ear of a rat. A second fiber optic bundle was also positioned near the same ear to detect the emitted fluorescent light and the other end was directed into the optics and electronics for data collection. An interference filter (IF) in the collection optics train was used to select emitted fluorescent light of the appropriate wavelength for the dye chromophore.
  • IF interference filter
  • Sprague-Dawley or Fischer 344 rats were used in these studies.
  • the animals were anesthetized with urethane administered via intraperitoneal injection at a dose of 1.35 g/kg body weight.
  • a 21 gauge butterfly with 12" tubing was placed in the lateral tail vein of each animal and flushed with heparinized saline.
  • the animals were placed onto a heating pad and kept warm throughout the entire study.
  • the lobe of the left ear was affixed to a glass microscope slide to reduce movement and vibration. Incident laser light delivered from the fiber optic was centered on the affixed ear. Data acquisition was then initiated, and a background reading of fluorescence was obtained prior to administration of the test agent.
  • the peptide-dye conjugate was administered to the animal through a bolus injection in the lateral tail vein, typically of 0.5 to 2.0 mL.
  • the fluorescence signal rapidly increased to a peak value.
  • the signal then decayed as a function of time as the conjugate cleared from the bloodstream.

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Abstract

Cette invention concerne des conjugués teinture-peptide qui conviennent pour l'imagerie médicale et la thérapie. Ces conjugués teinture-peptide comprennent plusieurs teintures cyanines avec une variété d'homologues bis- et tétrakis(acide carboxylique). Les petites dimensions de ces composés en comparaison du poids moléculaire plus élevé des agents pour imagerie permettent un apport plus favorable dans les cellules des tumeurs. Ces diverses teintures conviennent sur une plage allant de 350 à 1300 nm, la plage exacte dépendant de la teinture particulière. L'utilisation de diméthylsulfoxyde aide à maintenir la fluorescence de ces composés. Les molécules de cette invention sont utiles dans le domaine de l'imagerie médicale et de la thérapie, dans des applications endoscopiques permettant de détecter des tumeurs et d'autres anomalies et permettant des thérapies localisées, dans le domaine de l'imagerie photo-acoustique des tumeurs, pour la détection et la thérapie, dans le domaine de l'imagerie des tumeurs par sonofluorescence, pour la détection et la thérapie.
PCT/US2000/011060 1999-05-20 2000-04-26 Nouveaux bioconjugues cyanine et indocyanine pour application medicale Ceased WO2000071162A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE60042522T DE60042522D1 (de) 1999-05-20 2000-04-26 Cyanin- oder indocyaninefarbstoffe biokonjugate für biomedische anwendungen
DK00926343T DK1178830T3 (da) 1999-05-20 2000-04-26 Cyanin- og indocyaninfarvestof-biokonjugater til biomedicinske anvendelser
AT00926343T ATE435662T1 (de) 1999-05-20 2000-04-26 Cyanin- oder indocyaninefarbstoffe biokonjugate für biomedische anwendungen
CA2373475A CA2373475C (fr) 1999-05-20 2000-04-26 Nouvelle teinture de bioconjugues cyanine et indocyanine pour application biomedicale
JP2000619463A JP2003500367A (ja) 1999-05-20 2000-04-26 生物医学的適用のための新規なシアニンおよびインドシアニン染料バイオコンジュゲート
AU44888/00A AU4488800A (en) 1999-05-20 2000-04-26 Novel cyanine and indocyanine dye bioconjugates for biomedical applications
EP00926343A EP1178830B1 (fr) 1999-05-20 2000-04-26 Nouveaux bioconjugues cyanine et indocyanine pour application medicale

Applications Claiming Priority (4)

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US13506099P 1999-05-20 1999-05-20
US60/135,060 1999-05-20
US09/325,769 1999-06-04
US09/325,769 US6217848B1 (en) 1999-05-20 1999-06-04 Cyanine and indocyanine dye bioconjugates for biomedical applications

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WO2000071162A2 true WO2000071162A2 (fr) 2000-11-30
WO2000071162A3 WO2000071162A3 (fr) 2001-07-05

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AT (1) ATE435662T1 (fr)
AU (1) AU4488800A (fr)
CA (1) CA2373475C (fr)
DE (1) DE60042522D1 (fr)
DK (1) DK1178830T3 (fr)
ES (1) ES2329542T3 (fr)
PT (1) PT1178830E (fr)
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WO2005089813A3 (fr) * 2004-03-15 2006-08-17 Mallinckrodt Inc Contraste optique exogene avide de recepteur et agents therapeutiques
EP2181119A4 (fr) * 2007-07-20 2012-01-25 Ge Healthcare Bio Sciences Ab Marqueurs de point isoélectrique
EP2100621A1 (fr) 2008-03-10 2009-09-16 mivenion GmbH Conjugués de dendrimère de polyol de polyéther dotés de molécules effectrices pour le ciblage biologique
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EP1178830B1 (fr) 2009-07-08
US6217848B1 (en) 2001-04-17
WO2000071162A3 (fr) 2001-07-05
CA2373475A1 (fr) 2000-11-30
EP2058007A2 (fr) 2009-05-13
AU4488800A (en) 2000-12-12
DE60042522D1 (de) 2009-08-20
DK1178830T3 (da) 2009-10-12
PT1178830E (pt) 2009-09-02
JP2003500367A (ja) 2003-01-07
CA2373475C (fr) 2012-10-23
ATE435662T1 (de) 2009-07-15
ES2329542T3 (es) 2009-11-27
EP1178830A2 (fr) 2002-02-13
EP2058007A3 (fr) 2009-05-27

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